Controls
Seven control categories are built into the Illumina BeadChip system, six of which are being used by GPCF. These cover every aspect of an array experiment from the biological specimen, to sample labeling, to hybridization and signal generation. We automatically track performance of these controls and generate a report for each array.
Controls for the Biological Specimen (Housekeeping Controls): The intactness of the biological specimen can be monitored by housekeeping gene controls. These controls consist of probes to housekeeping genes, two probes per gene that should be expressed in any cellular sample.
Controls for Sample Labeling (RNA spike): These controls are NOT used at GPCF. These controls consist of four probes corresponding to artificial polyadenylated spike RNAs (Bacillus subtilis). The RNAs must be spiked into each sample immediately preceding the reverse transcription step. These spike RNAs are amplified and labeled in the same reaction as the sample, and thus act as tracers for reaction success.
Cy3-Labeled Hyb Controls: These controls consist of six probes with corresponding Cy3-labeled oligonucleotides present in the hybridization buffer. Following successful hybridization, they produce a signal independent of both the cellular RNA quality and success of the sample prep reactions. Target oligonucleotides for the Cy3 Hyb controls are present at three concentrations (low, medium, and high), yielding gradient hybridization response. |
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Low Stringency Hyb Control: This control category contains four probes, corresponding to the medium- and high-concentration Cy3 hyb control targets. In this case, each probe has two mismatch bases distributed in its sequence. If stringency is adequate, these controls yield very low signal. If stringency is too low, they yield signal approaching that of their perfect match counterparts in the Cy3 hyb control category. |
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High Stringency Hyb Control: This control consists of one probe corresponding to a Cy3-labeled oligonucleotide target. The probe/target sequences have a very high G+C content, and should thus hybridize even if hybridization stringency is too high. Hybridizations with too-high stringency are detected on the basis of a signal present from this control in the absence of signal from the other hybridization control probes. |
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Signal Generation Controls (Biotin Control): This category consists of two probes with complementary biotin-tagged oligonucleotides present in the Hyb E1 buffer. Successful secondary staining is indicated by a positive hybridization signal from these probes. |
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Negative Controls: This category consists of 800-1600 probes of random sequence selected to have no corresponding targets in the genomes. The mean signal of these probes defines the system background. This is a comprehensive measurement of background, representing the imaging system background as well as any signal resulting from non-specific binding of dye or cross-hybridization. We use the signals and signal standard deviation of these probes to establish gene expression detection limits. |
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